1. Field of the Invention
The present invention relates to a method for mass production of taxol from Taxus genus plant cell culture, more specifically, to a method for mass production of taxol by semi-continuous culture of Taxus genus plant cell with a high yield.
2. Description of the Prior Art
Taxanes are diterpene compounds containing a taxane skeleton. For example, taxol is famous as the first identified compound with a taxane ring which was isolated from the bark of the pacific yew, Taxus brevifolia, which is effective for the treatment of leukemia and cancer. Recently, it has been reported that: taxol is capable of curing approximately 30%, 50% and 20% of ovarian, breast and lung cancer patients, respectively, by inhibiting depolymerization of microtubules (see: E. K. Rowinsky et al., J. Natl. Cancer. Inst., 82:1247-1259 (1990)).
On the other hand, total chemical synthesis, semi-synthesis and extraction methods have been employed to prepare taxol.
The total chemical synthesis method, however, has not been practically applied in the art, since it requires very expensive chemical reagents and the yield is not so high, which can be expected from the complicated chemical structure of taxol.
The semi-synthesis method employing precursors such as 10-deacetylbaccatin III, has revealed some drawbacks since it essentially entails complicated and multiple steps of isolating and purifying the taxol precursors from Taxus genus plant and transforming the precursors to taxol.
In this regard, the extraction method by which taxol can be isolated from Taxus genus plants in a direct manner, has prevailed in the art, since it has the advantage of economy. However, the said method has proven to be less satisfactory in the sense that it essentially requires a large amount of yew trees to purify taxol, which finally gives rise to the serious environmental disruption.
Accordingly, the ability of total chemical synthesis, semi-synthesis and extraction method to supply taxol for world-wide chemotherapeutic use is not assured; and, there are strong reasons for exploring and developing alternative means of taxol production.
As a promising alternative to solve said problems, the cell culture method for taxol production has been proposed in the art.
The cell culture-based process for taxol production, unlike the prior art, has the following advantages as followings: first, taxol can be produced in a steady manner, regardless of fluctuation of supply of yew plants due to the damage by blight and harmful insects, etc.; secondly, cell cultures can be propagated in large bioreactors, from which taxol can be massively produced by manipulating culture conditions; thirdly, cell cultures produce a simpler spectrum of compounds compared to other prior art methods, considerably simplifying separation and purification; fourthly, a cell culture process can adapt quickly to rapid changes in demand better than the other methods; fifthly, a cell culture process can produce taxol as well as taxane precursors such as baccatin that can be converted to taxol.
Methods for producing taxol by utilizing cultured plant cells have been described in the art:
U.S. Pat. No. 5,019,504 discloses a method for producing taxol and its derivative utilizing cultured cells of Taxus brevifolia. However, the yield of taxol described therein is 1-3 mg/L, which is insufficient for industrial application. Besides, the production of taxol by the cell culture is unstable and even when a primary cell of high productivity can be obtained by selection, it is difficult to keep its content by subculturing (see: E. R. M. Wickremesine et al., World Congress on Cell and Tissue Culture (1992)).
U.S. Pat. No. 5,015,744 teaches a semi-synthetic method from baccatin III, which is a precursor in biosynthesis of taxol. By the use of the plant tissue culture, a raw material for the semi-synthetic process such as baccatin III can be produced, thus the plant tissue culture can also be utilized for taxol production by the above-mentioned semi-synthetic process.
WO 93/17121 offers a method for taxol production by cell culture of Taxus genus plant while changing composition of medium, growth rate, and production rate, etc. In case of Taxus chinensis, 24.1 mg/L of taxol can be obtained in 18 days of culture and the biomass doubles every 2.5 days.
All of these patents describes methods for mass production of taxol by employing batch culture; there is no teaching in said patents on, nor is there anticipated, semi-continuous culture of a taxol-producing cell line.
Under the circumstances, U.S. Pat. No. 5,407,816 describes that Taxus chinensis cells are inoculated to a nutrient medium to form a suspension which is, in turn, cultivated to form a suspension culture which is, in turn, subcultured in the other nutrient medium to form a producing culture, and which finally gives taxol and taxanes in a yield of 153 mg/L. The said method has considerably improved the productivity of taxol, however, it has been proven less satisfactory in the sense that it essentially requires so many different nutrient media whose compositions are so complicated, and high productivity can be realized under rather a limited growth condition.
Therefore, there is a continued need to develop a practical and simple method for taxol production, which is able to meet the requirement of high productivity which is a critical factor to determine whether it can be utilized in industrial applications or not.